Species specific antigen sequences for tick-borne relapsing fever (tbrf) and methods of use

ABSTRACT

Compositions and methods for the detection and identification of Tick-Borne Relapsing Fever  Borrelia  sp. antibodies.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.application Ser. No. 15/916,717, filed Mar. 9, 2018, the disclosure ofwhich is incorporated by reference herein in its entirety.

BACKGROUND

Borreliosis is caused by two groups of Borrelia, the B. borgdorferigroup and the Tick-Borne Relapsing Fever (TBRF) Borrelia group. It wasbelieved that the B. borgdorferi group was the only group that causedLyme-like symptoms in infected subjects. However, it has been learnedthat TBRF Borrelia also causes Lyme-like symptoms. TBRF Borrelia istransmitted by hard (Ixodes) and soft (Ornithodorus) ticks.

Borrelia bacteria that cause TBRF are typically transmitted to humansthrough the bite of infected “soft ticks” of the genus Ornithodorus.Soft ticks differ in two important ways from the more familiar “hardticks” (e.g., the dog tick and the deer tick). First, the bite of softticks is brief, usually lasting less than half an hour. Second, softticks do not search for prey in tall grass or brush. Instead, they livewithin rodent burrows, feeding as needed on the rodent (for example,squirrels, chipmunks and prairie dogs) as it sleeps.

The main symptoms of TBRF are high fever (e.g., 103° F.), headache,muscle and joint aches. Symptoms can reoccur, producing a telltalepattern of fever lasting roughly 3 days, followed by 7 days withoutfever, followed by another 3 days of fever. Without antibiotictreatment, this process can repeat several times.

Humans typically come into contact with soft ticks when they sleep inrodent-infested cabins. The ticks emerge at night and feed briefly whilethe person is sleeping. The bites are painless, and most people areunaware that they have been bitten. Between meals, the ticks may returnto the nesting materials in their host burrows.

There are several Borrelia species that cause TBRF, and these areusually associated with specific species of ticks. For instance, B.hermsii is transmitted by O. hermsi ticks, B. parkerii by O. parkeriticks, and B. turicatae by O. turicata ticks. Each tick species has apreferred habitat and preferred set of hosts.

Soft ticks can live up to 10 years. Individual ticks will take manyblood meals during each stage of their life cycle, and some species canpass the infection along through their eggs to their offspring. The longlife span of soft ticks means that once a cabin or homestead isinfested, it may remain infested unless steps are taken to find andremove the rodent nest.

Since TBRF can be caused by several species of Borrelia, tests need tobe comprehensive by including all known species. Also, identification ofthe Borrelia species can aid in identifying the host rodent foreradication. Currently, the standard for identification is byidentification of TBRF spirochetes in blood smears of a subjectpresenting symptoms consistent with TBRF. After obtaining the blood drawthe sample must be cultured for at least 24 hours to facilitateidentification. However, even early in the disease when spirochetes arehighest, positive identification is only made about 70% of the time.(See, www.cdc.gov/relapsing-fever/clinicians/index.html). Thus, priorart materials and methods result in a delay in diagnosis and provide arelatively low level of sensitivity and specificity.

Therefore, what is needed are new materials and methods suitable for theidentification of TBRF causative agents with decreased assay time andincreased sensitivity and specificity.

SUMMARY OF THE INVENTION

The present invention solves these problems in the prior art byproviding antigen-specific amino acid sequences for TBRF Borreliaspecific species. These novel amino acid sequences are used in assays toidentify TBRF specific Borrelia in samples from subjects suspected ofhaving TBRF. With the amino acid sequences of the present invention,identification of TBRF Borrelia in subject samples is performed withgreater speed, sensitivity and specificity than the prior art methods.The amino acid sequences of the present invention can be used indiagnostic and scientific assays. Examples of suitable assays areImmunoblots, ELISA (enzyme-linked immunosorbent assay), etc. The aminoacid sequences of the present invention can be used for the detection ofTBRF Borrelia specific T-cells (e.g., the IgXSPOT test; IGeneX, PaloAlto, Calif.). Further, and importantly, antigens encoded by the aminoacid sequences of the present invention can be used in vaccinationprotocols.

Thus, the present invention contemplates a composition comprising one ormore labeled and/or tagged and/or bound amino acid sequences, said aminoacid sequences consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

The present invention further contemplates that the sequences of thepresent invention, when bound, are bound to a substance selected fromthe group consisting of nitrocellulose, nylon, polyvinylidene difluoride(PVDF), magnetic beads and agarose.

The present invention further contemplates that the sequences of thepresent invention, when tagged, are tagged with an antibody withspecificity for said amino acid sequence.

Further, the present invention contemplates a method of detectingBorrelia antisera in a sample from a subject suspected of havingtick-borne relapsing fever (TBRF), the method comprising: providing abiological sample obtained from a subject suspected of having TBRF,mixing the biological sample with one or more of the labeled, taggedand/or bound amino acid sequences of the present invention and detectinga positive immunobinding reaction which indicates the presence of TBRFantisera in the sample.

The present invention further contemplates that two or more of thelabeled and/or tagged and/or bound amino acid sequences of the presentinvention are mixed with the biological sample. A sample is consideredpositive for TBRF if at least two amino acid sequences are detected. Inanother embodiment, a sample is considered positive for TBRF if at leastone amino acid sequences is detected.

The present invention further contemplates that the labeled and/ortagged and/or bound amino acid sequences are detected with anti-humanIgG antibody linked to a detectable moiety. The present inventioncontemplates that the detectable moiety may be selected from the groupconsisting of chromophores, radioactive moieties and enzymes. Thepresent invention contemplates that the detectable moiety may comprisealkaline phosphatase. The present invention contemplates that thedetectable moiety may comprise biotin.

The present invention further contemplates a method of detecting anddistinguishing various species of Borrelia in a sample from a subjectsuspected of having tick-borne relapsing fever (TBRF), said methodcomprising: providing a biological sample obtained from a subjectsuspected of having TBRF, mixing the biological sample with one or moreof the labeled and/or tagged and/or bound amino acid sequences of thepresent invention and detecting a positive immunobinding reaction whichindicates the presence of Borrelia in the subject, wherein detection ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 16 and/or SEQ ID NO: 17 indicates the presence of B. hermsii,in the subject; detection of SEQ ID NO: 3, SEQ ID NO: 8, SEQ D NO: 12and/or SEQ ID NO: 14 indicates the presence of B. miyamotoi in thesubject; detection of SEQ ID NO: 4, SEQ ID NO: 5; SEQ ID NO: 9 and/orSEQ ID NO: 15 indicates the presence of B. turcica in the subject;detection of SEQ ID NO: 6 and/or SEQ ID NO: 10 indicates the detectionof B. turicatae in the sample; and, detection of SEQ ID NO: 18, SEQ IDNO: 19 and/or SEQ ID NO: 20 indicates the detection of B. coriaceae inthe sample. The labeled and/or tagged and/or bound amino acid sequencesmay be detected with anti-human IgG antibody conjugated to a detectablemoiety. The present invention further contemplates that the labeledand/or tagged and/or bound amino acid sequences are detected withanti-human IgG antibody linked to a detectable moiety. The presentinvention contemplates that the detectable moiety may be selected fromthe group consisting of chromophores, radioactive moieties and enzymes.The present invention contemplates that the detectable moiety maycomprise alkaline phosphatase. The present invention contemplates thatthe detectable moiety may comprise biotin. The present invention furthercontemplates that a sample is considered positive for Borrelia if atleast one amino acid sequence is detected. The present invention furthercontemplates that a sample is considered positive for a specific speciesof Borrelia if at least one amino acid sequence identified with aspecific species is detected.

The present invention contemplates a method of detecting B. hermsii in asample from a subject suspected of having tick-borne relapsing fever(TBRF), said method comprising: providing a biological sample obtainedfrom a subject suspected of having TBRF, mixing the biological samplewith one or more of the labeled and/or tagged and/or bound amino acidsequences selected from the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and/or SEQ ID NO: 17of claim 1 and detecting a positive immunobinding reaction of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:16 and/or SEQ ID NO: 17 the detection of which indicates the presence ofB. hermsii in the sample.

The present invention contemplates a method of detecting B. miyamotoi ina sample from a subject suspected of having tick-borne relapsing fever(TBRF), said method comprising: providing a biological sample obtainedfrom a subject suspected of having TBRF, mixing the biological samplewith one or more of the labeled and/or bound amino acid sequencesselected from the group of SEQ ID NO: 3, SEQ ID NO: 8, SEQ D NO: 12and/or SEQ ID NO: 14 of claim 1 and detecting a positive immunobindingreaction of SEQ ID NO: 3, SEQ ID NO: 8, SEQ D NO: 12 and/or SEQ ID NO:14 which indicates the presence of B. miyamotoi in the sample

The present invention contemplates a method of detecting B. turcica in asample from a subject suspected of having tick-borne relapsing fever(TBRF), said method comprising: providing a biological sample obtainedfrom a subject suspected of having TBRF, mixing the biological samplewith one or more of the labeled and/or tagged and/or bound amino acidsequences selected from the group of SEQ ID NO: 4, SEQ ID NO: 5; SEQ IDNO: 9 and/or SEQ ID NO: 15 of claim 1 and detecting a positiveimmunobinding reaction of SEQ ID NO: 4, SEQ ID NO: 5; SEQ ID NO: 9and/or SEQ ID NO: 15 which indicates the presence of B. turcica in thesample.

The present invention contemplates a method of detecting B. turicatae ina sample from a subject suspected of having tick-borne relapsing fever(TBRF), said method comprising: providing a biological sample obtainedfrom a subject suspected of having TBRF, mixing the biological samplewith one or more of the labeled and/or tagged and/or bound amino acidsequences selected from the group of SEQ ID NO: 6 and/or SEQ ID NO: 10of claim 1 and detecting a positive immunobinding reaction of SEQ ID NO:6 and/or SEQ ID NO: 10 which indicates the presence of B. turicatae inthe sample.

The present invention contemplates a method of detecting B. coriaceae ina sample from a subject suspected of having tick-borne relapsing fever(TBRF), said method comprising: providing a biological sample obtainedfrom a subject suspected of having TBRF, mixing the biological samplewith one or more of the labeled and/or tagged and/or bound amino acidsequences selected from the group of SEQ ID NO: 18, SEQ ID NO: 19 and/orSEQ ID NO: 20 of claim 1 and detecting a positive immunobinding reactionof SEQ ID NO: 18, SEQ ID NO: 19 and/or SEQ ID NO: 20 of claim 1 whichindicates the presence of B. coriaceae in the sample.

The present invention further contemplates a composition comprising,consisting essentially of or consisting of one or more amino acidsequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, and an adjuvant.

The present invention further contemplates a nucleic acid sequenceencoding any one or more of the amino acid sequences of the presentinvention. Said nucleic acid sequence may be labeled.

The present invention further contemplates a method of detecting T cellsfrom a subject having or suspected of having tick-borne relapsing fever(TBRF), the method comprising: providing i) a sample comprising T cellsobtained from a subject suspected of having TBRF, ii) a cultureapparatus coated at least partially with anti-antibodies specific forone or more cytokines and iii) one or more sequences of claim 1; mixingthe blood sample with the one or more of the sequences of claim 1 in theculture apparatus, and detecting any production of the one or morecytokines by the subject's T cells. In one embodiment, theanti-antibodies that are specific for a cytokine are anti-IFNγantibodies and the cytokine is interferon gamma (IFNγ).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an Immunoblot using the TBRF Borrelia specific antigenicpeptide encoding amino acid sequences of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions and methods fordiagnosing, treating and vaccinating against infection by Tick-BorneRelapsing Fever causing Borrelia sp. The invention is based, in part, onthe discovery of species-specific amino acid sequences encodingantigenic peptides (which may also be referred to as peptide antigens orantigens in the art), as described below. Further, the present inventionprovides nucleic acid sequences encoding the amino acid sequences of thepresent invention. The nucleic acid sequences may be labeled.

The present invention provides for antigenic amino acid sequencesspecific for various Borrelia species. The amino acid sequences of thepresent invention encode antigenic peptides that have high specificityand/or sensitivity for the indicated species.

The present invention, in one aspect, is a composition comprising one ormore labeled and/or bound amino acid sequences, said amino acidsequences having 90%, 95%, 98%, 99%, 99.5% and/or 100% homology to SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20. Sequences less than 100% homologous may have deletions, additionsand or substitutions of the 100% homologous sequence. One of ordinaryskill in the art can easily determine if sequences less than 100%homologous can bind naturally or non-naturally occurring TBRF-relatedantibodies as well as the sensitivity and specificity of the antibody tothe modified sequences. In other words, one of ordinary skill in the artwill be able to identify sequences with significant homology to SEQ IDNOs: 1-20 of the present invention that give acceptable or equivalentresponses in the methods of the present invention without undueexperimentation, in view of the teachings of this specification.

The present invention, in one aspect, is a composition comprising one ormore labeled and/or tagged and/or bound amino acid sequences, said aminoacid sequences consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

With regard to the present invention, the phrase “a compositioncomprising one or more labeled and/or tagged and/or bound amino acidsequences, said amino acid sequences consisting of,” encompasses acomposition having the one or more of the recited sequences and, forexample, buffers, labels, etc. In other words, the sequence is limitedto the sequence or sequences given but the composition is not limited.The definition specifically excludes amino acids naturally contiguouswith a recited sequence being used as a label or tag as an element ofthe “composition comprising.”

In the context of the present invention, a “tagged” amino acid sequenceis an amino acid sequence that is attached to a detectable moiety.Non-limiting examples of such “tags” are natural and synthetic (i.e.,non-naturally occurring) nucleic acid and amino acid sequences (e.g.,poly-AAA tags), antibodies and detectable moieties such as labels(discussed below). Thus, the definitions of the phrases “labeled” and“tagged” may have overlap in that a tag may also, in some instances,function as a label. Further, tags useful with the present invention maybe linked to a label.

The amino acid sequences of the present invention, or any tags attachedto an amino acid sequence of the present invention, may be labeled withany suitable label known to one of ordinary skill in the art. Suchlabels may include, but are not limited to, biotin/streptavidin, enzymeconjugates (e.g., horseradish peroxidase (HRP), alkaline phosphatase(AP), glucose oxidase and β-galactosidase), fluorescent moieties (e.g.,FITC, fluorescein, rhodamine, etc.), biological fluorophores (e.g.,green fluorescent protein, R-phycoerythrin) or other luminescentproteins, etc. Any suitable label known to one of ordinary skill in theart may be used with the present invention.

Further, the amino acid sequences of the present invention may be“bound.” A “bound” amino acid sequence is an amino acid sequence thathas been immobilized in order to permit the use of the amino acidsequence in a biological test such as, for example, immunoassays. In thecontext of the present invention, a “bound” amino acid sequence is anamino acid sequence attached (e.g., covalently or non-covalently bound,etc.) directly or indirectly to a non-natural surface or substance.Further still, the “bound” amino acid sequences of the present inventionmay be attached, directly or indirectly, to a natural surface orsubstance, either of which is not naturally associated with the aminoacid sequence. Non-limiting examples of substances to which the aminoacid sequences of the present invention may be bound are nitrocellulose,nylon, polyvinylidene difluoride (PVDF), plastics, metals, magneticbeads and agarose (e.g., beads). Linking agents known to those ofordinary skill in the art may be used to aid or enhance binding of theamino acid sequences of the present invention to a surface or substance.

The present invention, in one aspect, is a composition comprising,consisting essentially of or consisting of one or more labeled and/ortagged and/or bound amino acid sequences, said amino acid sequencesconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19 and SEQ ID NO: 20 and up to five and up to ten additional aminoacids added to one or both of the 3′-prime and 5′-prime ends of thesequence, wherein said additional amino acids may or may not be thenaturally contiguous amino acids.

The amino acid sequences of the present invention may be naturaloccurring and isolated from a natural source. Further, the amino acidsequences of the present invention may be non-natural, syntheticsequences, such as sequences produced by recombinant technology orsequences synthesized by protein synthesizing apparatuses. As such, theamino acid sequences of the present invention may be isolated or may beproduced by recombinant technology, as is described and enabled in theliterature and in commonly referred to manuals such as, e.g., ShortProtocols in Molecular Biology, Second Edition, F. M. Ausubel, Ed., allJohn Wiley & Sons, N.Y., edition as of 2008; and, Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, 2001, and as is well known to oneof ordinary skill in the art. In one embodiment, the amino acidsequences of the present invention are made recombinantly in E. coli.

The amino acid sequences of the present invention may be tagged with anantibody with specificity for any of said amino acid sequences.Specificity for said amino acid sequence, i.e., antibody specificity, isthe property of antibodies which enables them to react preferentiallywith some antigenic determinants and not with others. Specificity isdependent on chemical composition, physical forces and molecularstructure at the binding site. Sensitivity is how strongly the antibodybinds to the antigenic determinant. One of ordinary skill in the art caneasily determine specificity and sensitivity of an antibody for aparticular amino acid sequence using standard affinity assays, such asimmunoblotting, Ouchterlony assays, titer assays, etc.

In another aspect, the present invention provides a method of quicklyand accurately detecting Borrelia antisera in a sample from a subjectsuspected of having tick-borne relapsing fever (TBRF). A subjectsuspected of having TBRF can be identified as having symptoms such as ahigh fever (e.g., 103° F.), headache, muscle and joint aches. Symptomstypically reoccur, producing a telltale pattern of fever lasting roughly3 days, followed by approximately 7 days without fever, followed byanother 3 days of fever. Without proper antibiotic treatment, thisprocess can repeat several times. Since the symptoms of TBRF can mimic,for example, viral flu-like symptoms, accurate diagnosis of TBRF isimportant for providing an effective treatment for the subject. Thepresent invention provides a quick and easy diagnostic test fordetecting the presence of antibodies specific for the causative Borreliaspecies, thereby satisfying the need for such a test.

The method of the present invention for detecting Borrelia antisera in asample from a subject suspected of having TBRF, may comprise, forexample, providing a biological sample (e.g., blood, saliva) obtainedfrom a subject suspected of having TBRF, mixing the biological samplewith one or more of the labeled and/or bound amino acid sequences of thepresent invention and detecting a positive reaction which indicates thepresence of TBRF antisera in the sample. The antisera may be detectedby, for example, immunoblotting, Elispot, ELISA, Western blotting or anyother appropriate immunoassay known to one of ordinary skill in the art.These techniques are known to one of ordinary skill in the art andprocedures can be found in common technical references. While similar,each of these techniques has its advantages and disadvantages. Othersuitable techniques may be known to those of skill in the art and areincorporated herein.

Briefly, Western blotting can involve separating proteins byelectrophoresis and then transferring to nitrocellulose or other solidmedia (e.g., polyvinylidene fluoride or PVDF-membrane and nylonmembrane), and is described in more detail below. Immunoblotting canalso involve applying proteins to a solid media manually or by machine.Preferably, the proteins are applied in straight lines or spots anddried, binding them to the solid support medium, e.g., nitrocellulose.The proteins used in an immunoblot can be isolated from biologicalsamples or produced by recombinant technology, as is well known by thoseof ordinary skill in the art. The bound proteins are then exposed to asample or samples suspected of having antibodies specific for the targetproteins.

With this procedure, a known antibody can be used to determine if aprotein is present in a sample, such as when the proteins of lysed cellsare separated by electrophoresis and transferred to the solid medium.Western blotting allows for the identification of proteins by size aswell as by specificity for a specific antibody.

Similarly, with a procedure called immunoblotting, known proteins can bebound to the solid medium and samples, such as samples from subjectssuspected of having an infection, can be tested for the presence ofspecific antibodies in the sample by contacting the bound protein withthe sample. An antibody that binds the target protein is usuallyreferred to as the primary antibody. A secondary antibody, specific forconserved regions of the primary antibody (for example, arabbit-anti-human IgG antibody may be used to detect primary humanantibodies) is used to detect any bound primary antibodies. Thesecondary antibody is usually labeled with a detectable moiety forvisualization. Non-limiting examples of suitable labels include, forexample, chromophores such as biotin, radioactive moieties and enzymessuch as alkaline phosphatase, etc. The use of these and other materialsfor the visualization of antibodies are well known to one of ordinaryskill in the art.

The Enzyme-Linked ImmunoSpot (ELISPOT) method can detect human T cellsthat respond to TBRFspecific antigens in vitro. In an ELISPOT assay, thesurfaces of PVDF membrane in a 96-well microtiter plate are coated withcapture antibody that binds, for example, anti-Interferon gamma (IFNγ)or other cytokine-specific antibody. During the cell incubation andstimulation step, the T cells isolated from patient whole blood areseeded into the wells of the plate along with aforementionedsequence(s), and form substantially a monolayer on the membrane surfaceof the well. Upon stimulation of any antigen-specific cells with one ormore of the sequences of the present invention they are activated andthey release the IFNγ, which is captured directly on the membranesurface by the immobilized antibody. The IFNγ is thus “captured” in thearea directly surrounding the secreting cell, before it has a chance todiffuse into the culture media, or to be degraded by proteases and boundby receptors on bystander cells. Subsequent detection steps visualizethe immobilized IFNγ as an ImmunoSpot; essentially the secretoryfootprint of the activated cell.

For a specific example of an ELISPOT test, each well of the plate iscoated with a purified cytokine-specific antibody specific for the testor cell being detected. Subject's (i.e., a subject suspected of havingTBRF) T cells are isolated and cultured in each well and stimulated withrecombinant antigens of one or more sequences of the present invention.TBRF-positive patient cells secrete cytokine in response to stimuli,which is captured by the antibody coated in the well and furtherdetected by ELISA.

ELISA assays are also used to detect antigens. The ELISA assay canpermit the quantification of a specific protein in a mix of proteins(for example, a lysate) or determine if a peptide is present in asample. Likewise, ELISA assays can be used to determine if a specificantibody is present by using a specific antigen as a target. As usedwith the present invention, target amino acid sequence(s) are attachedto a surface. Then, if present in the sample being tested, the reactiveantibody can bind to the antigen. A secondary antibody linked to anenzyme is added, and, in the final step, a substance containing theenzyme's substrate is added. The subsequent reaction produces adetectable signal, most commonly a color change in the substrate.

In one aspect of the method of the present invention, a positive resultis indicated when two or more of the labeled and/or bound amino acidsequences of the present invention are mixed with the biological sampleand when at least two amino acid sequences are detected. In anotheraspect of the invention, a positive result is indicated when at leastone of the labeled and/or bound amino acid sequences of the presentinvention are mixed with the biological sample and when at least oneamino acid sequence is detected.

In the method of the present invention, any primary antibody bound to apeptide encoded by an amino acid sequence of the present invention maybe detected with anti-human antibodies, such as IgG or IgM, used as thesecondary antibody conjugated to a detectable moiety. As discussedabove, the detectable moiety may be selected from the group consistingof chromophores, radioactivity moieties and enzymes or other detectablemoiety known to one of ordinary skill in the art. In one embodiment, thedetectable moiety comprises alkaline phosphatase. In another embodimentthe detectable moiety comprises biotin.

In another aspect of the invention, a method is provided for detectingand distinguishing various species of Borrelia in a sample. The samplemay be from a subject suspected of having TBRF. The method may comprise,for example, providing a sample, for example, a biological sampleobtained from a subject suspected of having TBRF and mixing orcontacting the biological sample with one or more of the labeled and/orbound amino acid sequences of the present invention. Amino acids may belabeled to confirm their presence if positive results are not obtainedin the assay. The detection of a positive immunobinding reactionindicates the presence of Borrelia in the sample, wherein detection ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 16 and/or SEQ ID NO: 17 indicates the presence of B. hermsii,in the sample; detection of SEQ ID NO: 3, SEQ ID NO: 8, SEQ D NO: 12and/or SEQ ID NO: 14 indicates the presence of B. miyamotoi in thesample; detection of SEQ ID NO: 4, SEQ ID NO: 5; SEQ ID NO: 9 and/or SEQID NO: 15 indicates the presence of B. turcica in the sample, detectionof SEQ ID NO: 6 and/or SEQ ID NO: 10 indicates the detection of B.turicatae in the sample and, detection of SEQ ID NO: 18, SEQ ID NO: 19and/or SEQ ID NO: 20 indicates the detection of B. coriaceae in thesample. The assay used may be any of the assays described elsewhere inthis specification, or as are known to one of ordinary skill in the art.

In a preferred embodiment of the invention, a sample is consideredpositive for Borrelia if at least two amino acid sequences are detected.A sample is considered positive for a specific species of Borrelia if atleast two amino acid sequences identified with a species are detected.In another embodiment of the invention, a sample is considered positivefor Borrelia if at least one amino acid sequence is detected. A sampleis considered positive for a specific species of Borrelia if at leastone amino acid sequence identified with a species is detected.

In another aspect of the invention, a method is provided for detectingand distinguishing various species of B. hermsii in a sample. The samplemay be from a subject suspected of having TBRF. The method may comprise,for example, providing a sample, for example, a biological sampleobtained from a subject suspected of having TBRF and mixing orcontacting the biological sample with one or more of the labeled and/orbound amino acid sequences of the present invention specific for B.hermsii. The detection of a positive immunobinding reaction of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 16 and/or SEQ ID NO: 17 indicates the presence of B. hermsii, in thesample. The assay used may be any of the assays described elsewhere inthis specification, or as are known to one of ordinary skill in the art.A sample is considered positive for B. hermsii if at least one aminoacid sequence is detected.

In another aspect of the invention, a method is provided for detectingand distinguishing various species of B. miyamotoi in a sample. Thesample may be from a subject suspected of having TBRF. The method maycomprise, for example, providing a sample, for example, a biologicalsample obtained from a subject suspected of having TBRF and mixing orcontacting the biological sample with one or more of the labeled and/orbound amino acid sequences of the present invention. The detection of,for example, a positive immunobinding reaction of SEQ ID NO: 3, SEQ IDNO: 8, SEQ D NO: 12 and/or SEQ ID NO: 14 indicates the presence of B.miyamotoi in the sample. The assay used may be any of the assaysdescribed elsewhere in this specification, or as are known to one ofordinary skill in the art. A sample is considered positive for B.miyamotoi if at least one amino acid sequence is detected.

In another aspect of the invention, a method is provided for detectingand distinguishing various species of B. turcica in a sample. The samplemay be from a subject suspected of having TBRF. The method may comprise,for example, providing a sample, for example, a biological sampleobtained from a subject suspected of having TBRF and mixing orcontacting the biological sample with one or more of the labeled and/orbound amino acid sequences of the present invention. The detection of,for example, a positive immunobinding reaction of SEQ ID NO: 4, SEQ IDNO: 5; SEQ ID NO: 9 and/or SEQ ID NO: 15 indicates the presence of B.turcica in the sample. The assay used may be any of the assays describedelsewhere in this specification, or as are known to one of ordinaryskill in the art. A sample is considered positive for B. turcica if atleast one amino acid sequence is detected.

In another aspect of the invention, a method is provided for detectingand distinguishing various species of B. turicatae in a sample. Thesample may be from a subject suspected of having TBRF. The method maycomprise, for example, providing a sample, for example, a biologicalsample obtained from a subject suspected of having TBRF and mixing orcontacting the biological sample with one or more of the labeled and/orbound amino acid sequences of the present invention. The detection of,for example, a positive immunobinding reaction of SEQ ID NO: 6 and/orSEQ ID NO: 10 indicates the detection of B. turicatae in the sample. Theassay used may be any of the assays described elsewhere in thisspecification, or as are known to one of ordinary skill in the art. Asample is considered positive for B. turicatae if at least one aminoacid sequence is detected.

In another aspect of the invention, a method is provided for detectingand distinguishing various species of B. coriaceae in a sample. Thesample may be from a subject suspected of having TBRF. The method maycomprise, for example, providing a sample, for example, a biologicalsample obtained from a subject suspected of having TBRF and mixing orcontacting the biological sample with one or more of the labeled and/orbound amino acid sequences of the present invention. The detection of,for example, a positive immunobinding reaction of SEQ ID NO: 18, SEQ IDNO: 19 and/or SEQ ID NO: 20 indicates the detection of B. coriaceae inthe sample. The assay used may be any of the assays described elsewherein this specification, or as are known to one of ordinary skill in theart. A sample is considered positive for B. coriaceae if at least oneamino acid sequence is detected.

In another aspect of the present invention the amino acid sequences ofthe present invention may be used as vaccines in vaccination protocols.Vaccination protocols are well known to one of ordinary skill in theart. Briefly, vaccination is the administration of antigenic material (avaccine) to stimulate an individual's immune system to develop adaptiveimmunity to a pathogen. Vaccines can prevent or ameliorate morbidityfrom infection.

Vaccines are typically administered with adjuvants. Adjuvants arecompounds and mixtures of compounds designed or found to aid instimulation of the immune system. For example an adjuvant is apharmacological or immunological agent that modifies the effect of otheragents. Adjuvants may be added to a vaccine to modify the immuneresponse by boosting it such as to produce a greater quantity ofantibodies and provide longer-lasting protection, thus minimizing theamount of injected foreign material. Adjuvants may also be used toenhance the efficacy of a vaccine by helping to modify the immuneresponse to particular types of immune system cells. There are differentclasses of adjuvants that can skew immune responses in differentdirections. For example, adjuvants may selectively skew the immuneresponse by preferentially activating T cells or B cells depending onthe purpose of the vaccine. Adjuvants may also be used in the productionof antibodies from immunized animals. For example, rabbit anti-human IgGantibodies may be produced by inoculating a rabbit with conservedportions of a human IgG antibody in combination with an adjuvant. Themost commonly used adjuvants include aluminum hydroxide, paraffin oiland Freud's complete and incomplete adjuvant.

Although not fully understood adjuvants are believed to apply theireffects through different mechanisms. Some adjuvants, such as alum,function as delivery systems by generating depots that trap antigens atthe injection site, providing a slow release that continues to stimulatethe immune system. Other adjuvants augment or stimulation the hostimmune response by providing a general boost to the immune system.

In this regard, another aspect of the present invention is a compositioncomprising or consisting essentially of one or more amino acidsequences, said amino acid sequences having 90%, 95%, 98%, 99%, 99.5%and/or 100% homology selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 and anadjuvant. The adjuvant may be any adjuvant known to one of ordinaryskill in the art suitable for use in humans. Combinations two or moreadjuvants may be used. One of ordinary skill in the art will be able toidentify sequences of the present invention that give acceptable orequivalent immunological responses without undue experimentation. Bydefinition, with regard to the phrase “consisting essentially of,”agents other than the recited sequences and adjuvant are not consideredessential in the context of the present invention.

Isolated nucleic acid sequences, including polynucleotides andoligonucleotides, encoding the amino acid sequences of the presentinvention, and portions thereof, may be expressed in cultured cells toprovide isolatable quantities of peptides displaying biological (e.g.,immunological) properties of the antigenic peptide encoded by the aminoacid sequences of the present invention. Because of redundancy of thegenetic code, multiple nucleic acid sequences may be suitable for theproduction of the peptide sequences of the present invention. One ofordinary skill in the art will be able to determine one or more nucleicacid sequences for the production of the amino acid sequences of thepresent invention. The nucleic acid sequences encoding the amino acidsequences of the present invention may be labeled by any suitable labelknown to one of ordinary skill in the art.

In this regard, nucleic acid sequences of suitable for the production ofthe amino acid sequences of the present invention may be substantiallyhomologous to the naturally occurring sequences. Substantial homology ofa nucleic acid sequence means either that (a) there is greater thanabout 65%, typically greater than about 75%, more typically greater thanabout 85%, preferably greater than about 95%, and more preferablygreater than about 98% homology, most preferably 99% with the naturallyoccurring sequence or (b) the homologous nucleic acid sequence willhybridize to the compared sequence or its complementary strand understringent conditions of the temperature and salt concentration. Thesestringent conditions will generally be a temperature greater than about22° C., usually greater than about 30° C. and more usually greater thanabout 45° C., and a salt concentration generally less than about 1 M,usually less than about 500 mM, and preferably less than about 200 mM.The combination of temperature and salt concentration is more importantin defining stringency than either the temperature or the saltconcentration alone. Other conditions which affect stringency include GCcontent of the compared sequence, extent of complementarity of thesequences, and length of the sequences involved in the hybridization, aswell as the composition of buffer solution(s) used in the hybridizationmixture. These and other factors affecting stringency are well describedin the scientific and patent literature. One of ordinary skill in theart will be able to determine suitable conditions for determining thehomology of the nucleic acid sequences encoding the antigenic peptidesof the present invention.

Further, homologous nucleic acid sequences may be determined based onthe nature of a nucleotide substitution in the nucleic acid sequence.For example, conservative nucleotide substitutions will be toleratedbetter and, therefore, can be more numerous in a particular nucleic acidsequence than non-conservative nucleotide substitutions. One or ordinaryskill in the art will be able to determine the suitable number andlocation of substitutions that may be allowed in a nucleic acid sequencethat encodes an amino acid sequence of the present invention withoutadversely affecting the species specificity of the encoded antigenicpeptide, without undue experimentation.

The present invention will now be described in view of the following,non-limiting, exemplification.

EXEMPLIFICATION Example 1

Borreliosis is caused by two groups of Borrelia, B. burgdorferi groupand the Tick-Borne Relapsing Fever (TBRF) Borrelia group. It wasbelieved that B. burgdorferi group is the only group that causesLyme-like symptoms. However it is now known that TBRF Borrelia can alsocause Lyme-like symptoms. TBRF Borrelia can be transmitted by hard(Ixodes) and soft (Ornithodorus) ticks.

TBRF ImmunoBlot

The TBRF Borrelia immunoblot of the present invention is designed todetect antibodies to TBRF Borrelia species [including, but not limitedto Borrelia miyamotoi, B. hermsii, B. turicatae and B. coriaceae]specific antigens in human serum. For diagnostic purposes, immunoblottest results may be used in conjunction with clinical symptoms and otherevidence available to the diagnosing physician.

The TBRF Borrelia ImmunoBlot Test is a qualitative immunoblot assay thatdetects antibodies directed against TBRF associated Borrelia species insera of patients suspected of having TBRF Borrelia infection.Recombinant TBRF Borrelia antigens (protein sequences listed below) wereapplied as straight lines onto nitrocellulose strips, where they bind.The strips were then be used in the TBRF Borrelia ImmunoBlot Test.

Species specific Borrelia amino acid sequences of the present invention:

B. hermsii - BipA-1H [SEQ ID NO: 1]MSESNWEIDEPGSVQDIRNSVASELQKPENIGQRGKSVGKEVGKDAAASGEGAVVAVGSKQNTLQNSENSSQEGAGSALQKPGDSPQKGVASQEGTNGALQGVVAAGGVSVGGSGVGAAASDGNSSSSQEAESVDLKNVLADSHGVGASNLNIKAEGDISTGHGTEGVIASGDLTNTIITSGVTAAASPAIVSGDERGVAAIKDSIAVVLETQKEAQKETEKETENVKFAVLGTVKKIVDGVADGIANVIEYGLENGID B. hermsii - BipA-2H [SEQ ID NO: 2]MNKVKDGIKTASKGAETFVTAAMQGAGAVLGSLQDVGNFVMDATLSMGDMFAGIKSDSDVSSANSGMTVNSGMTVSLSSNETQVIGHLEEYLKSAIKVNGNESSRQSKLENGRQKFFAWLREKDTDFSKRKELVQAMQRVYNFIKEKSSNSRELQTWVLGVVGDDDTVVDVDRDDELNSDVEIDFLIKKTLSSRDYSGFAVSLLFQALADTLYDAENDRDKPEEQIFKDLKAVFSDNAGEDKGFGEFKSIIED KSQAB. miyamotoi - BipA [SEQ ID NO: 3]MAKAGNFDEVLDLDGDDEEAEIDEIEQNLSEGVDGNIDQNPGNVDGHIVLVDAQGVSVPTVVEHSAVSSSVERRVVTVGQNVESRRLTKVERLEKYLESAIKSGGKLNEQQRRLKNGKQMLFTWLNEDANASKRAELEQDMQKLYGLIKESITDSSSQFSSRDGEYSDEAIDGLLNSLFSSSFDNTFALDLFFQALLNTLCDFKKDNCKDEEVIFADIRKVFSDESDTKGHFGYLKSKLKDELVISDEGDYGED EDFEEDB. turcica - BipA1 [SEQ ID NO: 4]MEEEASIESEAMPLDEAKISSLDKETVPAVQADGTPEQIVASVGLEGEESKKFEHLKGSLGDAIKVNGKGEEKRKENEERQKEFFDWLDKNDPDLSKRKELAELMKKVYGLLKEHAQNSEQIKSFVEGTPKDENVKKIGVTSARDIKTDEQVEALIKAVLGHSEESGTNLSLFFQKLGDAFGTEDGESQKSNEKILEELKRVCESSDEIKKLKEDLKIEEKVQS B. turcica - BipA2 [SEQ ID NO: 5]MNIDAVADLLADQQEAASTGLKDASTGLKDTSTGLSKLDKKEKKVSSLKETLENSSNVLYESSSPTKTRQEEFFKWLEENDSDYSKRKQLEESMDKVFSLIKDSASSSTEIKEIIAKGQSDAGIIKAGIKTADDIKTDEQVDALVKFVTGTGDDLDLESGSSSIKAFFGTLAEVFDDDLNDVMTDKKGQKRGHDKVFEDLKKVFSEDSDGPFDILKDALKQALKNN B. turicatae - BipA-1H [SEQ ID NO: 6]MSTSYWSVDNDGFVQGTKSFVDSPLRKPDRFDQEVSAGGKKIEKAVSRNLRVAGGQRQGIADDGIGVAGVREAGGVLKDAGNAVQRDINGSGEGIKNDVIQNPEGVGVQVAVGSADTGADSGQEAGKVFQNLGDTGTQSIQRVVSSSDLNSDLGVGSKDGISTNGMSTNHVTENENSINSITSTSSGLNTALQMAGTSTRTSGYEGEITTNTQDRTFVETGTQDSKAQYSDFSDQDIRDKVLGSVVGGVVB. hermsii - GlpQ-s [SEQ ID NO: 7]MASMTGGQQMGRGSEKMSQNQKSPLVIAHRGASGYLPEHTLEAKAYAYALGADYLEQDIVLTKDNIPVIMHDPELDTTTNVAKLFPERARENGRYYSVDFTLDELKSLSLSERFDPASRNPIYPNRFPLNEHDFKIPTLEEEIQFIQGLNKSTGRNVGIYPEIKKPLWHKQQGKDISKIVIEILNKYGYKSKEDKIYLQTFDFDELKRIREELGYQGKLIMLVGENDWDEAPTDYEYIKSEEGMAEVAKYSDGIGPWIPQIIIEGKITDLTSLAHKHNMEVHPYTFRIDALPSYVKDADELLDLLFNKAKVDGIFTDFTDTVVNFITKIKPKGE B. miyamotoi - GlpQ-s [SEQ ID NO: 8]MASMTGGQQMGRGSEMGENKKSPLIIAHRGASGYLPEHTLEAKAYAYALGADYLEQDIVLTKDNIPVIMHDPEIDTTTNVAQLFPNRARENGRYYATDFTLTELKSLNLSERFDPENKKPIYPNRFPLNEYNFKIPTLEEEIQFIQGLNKSTGKNVGIYPEIKKPFWHKQQGKDISKIVIEILNKYGYKSKEDKIYLQTFDFDELKRIRKELGYQGKLIMLVGENDWNEAPTDYEYIKSEEGIAEVAKYSDGIGB. turcica - GlpQ [SEQ ID NO: 9]MASMTGGQQMGRGSKVSMNKALPLVIAHRGASGYLPEHTLEAKAFAYALGAHYLEQDIVLTKDDIPIIMHDPEIDTTTNVAEIFPERARKDGRYYSVDFTLRELKSLKLSERFDPKTGKPIYPNRFPLNEYNFKIPTLEEEIQFIQGLNKSTGRNVGIYPEIKKPFWHKQQGKDISKIVIEMLNKYGYKSKEDKIYLQIFDFDELKRIREELGYKGKLVMLIGENDWNEAPTDYEYIKSEEGIAEVAKYSDGIGPWIPQVIIDGKVTGLTSLAHKHKMEVHPYTMRIDALPSYVKDANELLNLLFNKAKVDGVFTDFPDVVLGFIR KB. turicatae - GlpQ [SEQ ID NO: 10]MASMTGGQQMGRGSEKMSMTNKKPPLIIAHRGASGYLPEHTLEAKAFAYALGADYLEQDIVLTKDNVPIIMHDPELDTTTNVAKLFPERARENGRYYSVDFTLDELKSLSLSERFDLETRKPIYPNRFPLNEYNVKIPTLEEEIQFIQGLNKSTGRNVGIYPEIKKPLWHKQQGKDISKIVIEILNKYGYKSKEDKIYLQTFDFDELKRIREELGYQGKLIMLVGENDWDEAPTDYEYIKSQEGMTEVAKYADGIGPWIPQIIIDGKITDLTSLAHKYNMEVHAYTFRIDSLPSYVKDANELLDLLFNKAKIDGLFTDFTDTVVKF VKQB. hermsii - BpcA [SEQ ID NO: 11]MASMTGGQQMGRGSSDANLLKTLDNNQKQALIYFKDTLQDKKYLNDLTASQKNFLDDLEKNKKDPGLQDKLKKTLSSEYDGSQFNKLLNELGNAKVKQFLQQLHIMLQSIKDGTLTSFSYANFKDLQTLEQKKERALQYINGRLYVEYYFYINGISNADNFFESVMQLLET B. miyamotoi - BpcA[SEQ ID NO: 12]MLDHNLQPNKINNIISSLDSNQKQALIFFKNLVKNKQYSKDLEQASKSYLENLKEKNNQNLNLQNKLNQGLNCDYDDSKIEKLFDQLGNDKMKKFLQQLHLMLKSINDGTLISFSSSNFRDTTTLSQKKEKALEYIKSQLYIEFYFHSNDISDTEFFFQRTIALLETQN B. hermsii - P41[SEQ ID NO: 13]MRNNSINATNLSKTQEKLSSGHRINRASDDAAGMGVAGKINAQIRGLSQASRNTSKAINFIQTTEGNLNEVERVLVRMKELAVQSGNGTYSDADRGSIQIEIEQLTDEINRIADQAQYNQMHMLSNKSAAQNVKTAEELGMQPAKINTPASLAGSQASWTLRVHVGANQDEAIAVNIYASNVANLFAGEGAQAAPVQEIGQQEEGQAAPAPAAAPAQGGVNSPINVTTAVDANMSLAKIEGAIRMVSDQRANLGAFQNRLESIKDSTEYAIENLKASYAQIKDATMTDEVVASTTHSILTQSAMAMIAQANQVPQYVLS LLRB. miyamotoi - P41 [SEQ ID NO: 14]MRNNGINAANLSKTQEKLSSGYRINRASDDAAGMGVAGKLNSQIRGLSQASRNTSKAINFIQTTEGNLNEVEKVLVRMKELAVQSGNGTYSDSDRGSIQIEIEQLTDEINRIADQAQYNQMHMLSNKSAAQNVKTAEELGMQPAKINTPASLAGSQASWTLRVHVGANQDEAIAVNIYAANVANLFNGEGAQAAPAQEGAQQEGVQAVPAPAAAPVQGGVNSPINVTTAIDANMSLSKIEDAIRMVTDQRANLGAFQNRLESVKASTDYAIENLKASYAQVKDAIMTDEIVASTTNSILTQSAMAMIAQANQVPQYVLS LLRB. turcica - P41 [SEQ ID NO: 15]MRNNGINASNLSKTQEKLSSGYRINRASDDAAGMGVAGKINAQIRGLSQASRNTSKAINFIQTTEGNLNEVEKVLVRMKELAVQSGNGTYSDADRGSIQIEIEQLTDEINRIADQSQYNQMHMLSNKSAAQNVKTAEELGMQPAKINTPASLSGAQASWTLRVHVGANQDEAIAVNIYAANVPNLFAGEGAQTAAAAPAQAGTQQEGAQEPAAAAAPAQGGVNSPVNVTTTVDANMSLAKIENAIRMISDQRANLGAFQNRLESIKNSTEYSIENLKASYAQIKDATMTDEIVSSTTNSILTQSAMAMIAQANQVPQYVL SLLRB. hermsii - BmpA10 [SEQ ID NO: 16]MSKAGSGLTKIAILVDGTFDDESFNGSAWKGAKKVEKEFGLEIMMKESNANSYLADLESLKNNGSNFLWLIGYKFSDFAIIAALENPESKYVIIDPVYESDLVIPENLSAITFRTEEGAFLVGYIAAKMSKTGKIGFLGGFDDVVVNTFRYGYEAGAIYANKHINIDNKYIGNFVNTETGKNMANAMYAEGVDIIYHVAGLAGLGVIESARDLGDGHYVIGVDQDQSHLAPDNVITSSIKDIGRVLNIMISNYLKTNAFEGGQVLSYGLKEGFLDFVKNPKMISFELEKELDDLSEGIINGKIIVPNNERTYNQFMRKILB. hermsii - OspC10 [SEQ ID NO: 17]MFLFISCNNGGPELKGNEVAKSDGTVLDLSKISTKIKNAGAFAASVQEVATLVKSVDELASAIGKKIKEDGTLDTLNNKNGSLLAGAFQVILTVEAKLKELEKQDGLSVELRAKVTSAKSASSGLVNKLKGGHAELGIEGATDENAQKAIKKDNGDQSKGAEELGKLNTAIGALLSAANDAVEAAIKELTAAPAKPATPAKP B. coriaceae - GlpQ [SEQ ID NO: 18]MKSMKPKLLMLIINIFLIISCQNEKVSMNEKSPLIIAHRGASGYLPEHTLEAKAYAYALGADYLEQDIVLTKDNVPIIMHDSELDTTTNVAKLFPERARENGKYYAVDFTLDEIKSLSISERFDPETREPIYPNRFPLNEYNFKIPTLEEEIQFIQGLNKSTGKNVGIYPEIKKPFWHKQQGKDISKIVIEILNQYGYKSKEDKIYLQTFDFDELKRIREELGYQGKLIMLVGENDWNEAPTDYEYIKSQEGMTEVAKYADGIGPWISQIIIDGQVTDLISLAHKHNMEVHPYTFRIDALPSYVKDANELLDLLFNKAQVDGIFTDFVDKAMEFVKKB. coriaceae - BIPA [SEQ ID NO: 19]MKSFSVFILFLSTFTLSCKFYDTANASDLQADGDKFSQGFSSFNDILPFSDLKINKDVSTGSLKAQGTSSIKGDEKKGKGTSKDPIKDQEASGLKGVGVAGAGAKSFGDDGKKEEVVSKDSLKNEGTSGTAEVLKVSKEVEVAGVDTAKPAGGNGEEVASISENYLQNQETLVAQGAGVGSVGDAIGDRSLFFKNTDSNNAEQVVATEDLLVGASEGVNTSDLGLKVAIPTDHVRGDVVATETQNAEKKGDKTQNTELASLDIKDNITVNVVDGTKININKNSSNTNESINVTKDGVNTVIKGVETSIKTADGKVVVKKRTLKKGLKKKNSKKQASKSKTPEAAVVGNKKNVDTNMSSVIGLDSEALGKDKNIDLDSKSDETYVIERVEKLAKYLQSAIKINGKKVEEQDKLEAGRQKFFEWLSKNDTDLLKRKALVQDLQKIYDLMKDKIADSTELQDWFQIVSDDIGDEETNIIDVESYYELSSDTEIDFLLERTLEDENYSGFSISLFMQALADTLYDIQNDSHKSGEEILQELKRVFDDTFYKIRGFEEFKSQIAAED B. coriaceae - BPCA[SEQ ID NO: 20]MKLTKKYLLAVLLLSLINCDLLSKNKILTSHLLNTLDNNKKEALVTFKNLLQDKSHLEYLKSEQAKMLTNFTEDDGIEQPHLQEKLKGTLSSEYNENQLNQLFSELGYEKTKQFLDNLHKMLQAIKDGTLRAFHDSSSFKDYNTTLEAKKAEALSSVKKELYVQYYFYINDLQTADDFFVLTRNHLMIFKNNL

ImmunoBlotting:

Immunoblotting is well known to one of ordinary skill in the art. Withregard to the present invention, to perform the TBRF Borrelia ImmunoBlottest, patient serum was incubated with TBRF Borrelia ImmunoBlot strips,produced as described above, in each trough of an incubation tray. Ifspecific antibodies to TBRF Borrelia antigens were present in a sample,they were bound to the corresponding antigen bands. After washing awayunbound antibodies, the bound TBRF Borrelia specific antibodies weredetected with alkaline phosphatase (AP) conjugated goat or rabbitanti-human IgG or IgM antibody. After removing the unbound conjugatedantibody, the strips were incubated with BCIP/NBT, an AP chromogenicsubstrate. A dark purple colored precipitate developed on theantigen-antibody complexes. Bands were visualized and scored forintensity relative to the positive, negative and calibrator(calibration) controls.

Example 2

Specificity of TBRF Borrelia ImmunoBlot

Method:

TBRF ImmunoBlot strips were tested with rabbit anti-TBRF Borrelia serumsamples and Borrelia burgdorferi serum samples. Rabbit antibodies to thefollowing Borrelia species were tested: B. hermsii, TBRF Borreliaspecies, B. coriaceae, B. burgdorferi B31, B. burgdorferi 297, B.californiensis, B. afzalii, B. garinii, B. spielmanii and B. valensiana.

Result Summary:

As shown in FIG. 1, in the columns numbered 1-3 under the heading“Relapsing Fever Borrelia species,” antibodies to B. hermsii, TBRFBorrelia sp., and B. coriaceae were detected. In the columns numbered1-7 under the heading “Lyme Disease Borrelia species,” only antibodiesto 41 kDa were detected with the rabbit anti-B. burgdorferi specificserum samples. The numbers refer to the antibodies denoted in the Figurekey.

Conclusion:

Based on the data presented above, the TBRF ImmunoBlot is very specificfor the detection of TBRF Borrelia specific antibodies.

Example 3

TBRF Borrelia ImmunoBlots Validation Study—Clinical Sensitivity andSpecificity

A total of 171 patient samples were tested as per TBRF BorreliaImmunoBlot IgM and IgG protocols to determine clinical sensitivity andspecificity. The following patient samples (Table 1) were tested as perTBRF Borrelia Immunoblot IgM and IgG test protocols. The ImmunoBlotswere read by in-house criteria. The ImmunoBlot (IgM or IgG) wasconsidered positive if 2 of the following bands were present: 21-23, 41,70-75 kDa and GlpQ. If only one of the following bands is present, theImmunoBlot was considered boarder-line positive: 21-23, 70-75 kDa andGlpQ. Results are summarized below in Tables 2a, 2b and 2c.

TABLE 1 List of Patient Samples Tested Study Set # Source Samples n Set1 In-house 34 samples tested by TBRF Western blots 34 (Lyme-likesymptoms, most negative by Lyme Western Blot and Lyme ImmunoBlots) Set 2In-house 10 samples - 2 samples/patient collected at 20 different timepoints (Negative by Lyme Western and ImmunoBlots) Set 3 CDC CDC -Specificity Samples (Provided Blinded 50 by CDC) Set 4 NY SpecificitySamples (purchased from NY 25 biologies Biologies) Set 5 In-houseAutoimmune and Allergy patient samples 42 (Previously left over PTsamples) Total Samples 171

TABLE 2a Results: Overall Summary. Table 2: Clinical Sensitivity -Overall Summary (see Table 2a and 2b) Lyme ImmunoBlot TBRF ImmunoBlotIgM &/ IgM &/ Sample Size Result IgM IgG or IgG IgM IgG or IgG Set 1 34Positive 2 1 2 15 8 18 Negative 32 33 32 19 26 16 Set 2 10 Positive 0 00 6 3 7 (old) Negative 10 10 10 4 7 3 10 Positive 0 0 0 8 5 10 (new)Negative 10 10 10 2 5 0 Total 54 Positive 2 1 2 29 16 35 Negative 52 5352 25 38 19

TABLE 2b Performance of TBRF Immunoblot on Clinical Samples Set 1:Patients with Lyme-like symptoms Patients Samples IgM IgG IgM and/orwith antibodies to: n (+) (+) IgG (+) Patients negative for Lyme 30 15 918 Bartonella 2 0 0 0 Ehrlichia 2 0 0 0 Total Positive 15 9 18 TotalNegative 16

To see how a patient's response immunologically to infection two sampleswere collected from each of 10 patients (including patients with ahistory of a tick bite and TBRF PCR positives). Results are presentedbelow in Table 2c.

TABLE 2c Performance of TBRF Immunoblot on 10 Positive Clinical SamplesSet 2: Patients with Lyme-like Symptoms, negative by Lyme ImmunoBlotsand Lyme Western blots (10 patient samples collected at 2 time points)IgM IgG IgM and/or Patient Samples n (+) (+) IgG (+) 1st collection 10 63 7 4 7 3 2nd collection 10 8 5 10 (6 weeks to 2 years latter) 2 5 0

Sensitivity:

Based on the data presented above for patients with Lyme-like symptoms,of the 44 patients (total 54 samples) with Lyme-like symptoms, 2patients had antibodies to B. burgdorferi and TBRF Borrelia; 29 patientshad antibodies to TBRF Borrelia. Four patients with other tick-bornediseases were negative by TBRF ImmunoBlot. Two sets of serum sampleswere collected from 10 patients, to see patient's immune response toinfection. We collected two samples at different time points from 10patients (including patients with a history of a tick bite and/or TBRFPCR positive samples). The second sample was collected 6 weeks to 2years after the first sample (See Table 2b and Table 2c for detailedresults). As shown above, 7 patients (4 IgM (+), 1 IgG (+) and 2 withIgM and IgG) were positive initially. When the second sample was testedall patients were positive, 5 IgM (+), 2 IgG (+) and 3 IgM and IgG (+).

Specificity:

Based on the data presented below in Table 3, the specificity of theTBRF Borrelia ImmunoBlot was 95.0% for IgM and 97.5% for IgG.

Conclusion:

The specificity of TBRF ImmunoBlot was 95.0% for IgM and 97.5% for IgG.With 10 patients (Set 2), we demonstrated that the immune responsevaried between individuals.

The results show that the TBRF ImmunoBlots can be used in to detect TBRFBorrelia-specific antibodies in patients suspected of TBRF Borreliosis.The results can be used by the physician in conjunction with patienthistory and symptoms. Detailed results are summarized below in Table 3.

TABLE 3 Clinical Specificity - Overall Summary IgM IgG IgM and/or SourceSample Types n (+) (+) IgG (+) 50 CDC Endemic Controls 10 0 0 0 SamplesFibromyalgia 5 0 0 0 (n = 50, Mononucleosis 5 0 0 0 Set 3) MultipleSclerosis 5 1 0 1 Non-endemic Controls 10 0 0 0 Periodontitis 5 0 0 0Rheumatoid Arthritis 5 1 0 1 Syphilis 5 0 1 1 IgM IgG IgM and/orAntibodies to: n (+) (+) IgG (+) New York Rapid Plasma 8 1 1 2 BiologiesReagin (RPR) (n = 25, Epstein-Barr 4 0 0 0 Set 4) virus (EBV) Human 4 00 0 immunodeficiency virus 1 (HIV-1) Cytomegalovirus 5 0 1 1 (CMV)Autoimmune Antinuclear 5 0 0 0 and Allergy antibody (ANA+) (n = 42,Antinuclear 4 0 0 0 Set 5) antibody (ANA−) DNA (+) 1 0 0 0 Rheumatoid 92 0 2 factor (+) Rheumatoid 8 0 0 0 Factor (−) IgG (+) 13 1 0 1 Spec.IgE (+) 4 0 0 0 Spec. IgE (−) 2 0 0 0 In-house Bartonella 2 0 0 0 (n =4, henselae Set 1) Human Granuloytic 2 0 0 0 Ehrlichia False Positive 63 9 True Negative 121 115 118 112 Specificity 95% 97.5% 93%

We claim:
 1. A composition comprising one or more labeled and/or taggedand/or bound amino acid sequences, wherein the one or more labeledand/or tagged and/or bound amino acid sequences consist of amino acidsequences selected from the group consisting of SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, and SEQ ID NO:
 20. 2. The composition of claim 1,wherein said amino acid sequences are bound to a substance selected fromthe group consisting of nitrocellulose, nylon, polyvinylidene difluoride(PVDF), magnetic beads and agarose.
 3. The composition of claim 1,wherein each of said one or more amino acid sequences are tagged with anantibody with specificity for said amino acid sequence.
 4. A method ofdetecting antibodies to one or more of B. hermsii, B. turicatae, B.miyamotoi, B. turcica, and B. coriacae amino acid sequences in a samplefrom a subject suspected of having tick-borne relapsing fever (TBRF),said method comprising: providing a biological sample obtained from asubject suspected of having TBRF; mixing the biological sample with oneor more of the labeled and/or tagged and/or bound amino acid sequencesof claim 1; and detecting a positive immunobinding reaction whichindicates the presence of TBRF specific antibodies in the sample.
 5. Themethod of claim 4, wherein two or more of the labeled and/or taggedand/or bound amino acid sequences are mixed with the biological sampleand a sample is considered positive for TBRF if at least two amino acidsequences are detected.
 6. The method of claim 4, wherein the labeledand/or tagged and/or bound amino acid sequences are detected withanti-human IgG or anti-human IgM antibody linked to a detectable moiety.7. The method of claim 6, wherein said detectable moiety is selectedfrom the group consisting of chromophores, radioactive moieties andenzymes.
 8. The method of claim 6, wherein said detectable moietycomprises alkaline phosphatase.
 9. The method of claim 6, wherein saiddetectable moiety comprises biotin.